The cyclic interaction between actin and myosin is involved in many of the motile processes in cells. While the atomic structures of the contractile proteins are known, the structural changes in the actomyosin complex involved in force generation is not yet resolved. Our goal is to understand the structural basis of contraction and its regulation in skeletal muscle. X-ray diffraction, one of our approaches, is one of the few non-invasive techniques that allow direct observation of structural changes in fully functioning muscle fibers. Currently major efforts are concentrated on studying the structures of the intermediate states of the actomyosin ATP hydrolysis cycle. Only by characterizing the structures of such states, structural transitions involved in force generation might be understood. Our previous studies (J. Physiol. 1993) on radial elasticity of muscle fibers indicated that the molecular structure of attached cross-bridges with MgADP may differ from the rigor (ATP-free) cross-bridge. Preliminary two-dimensional X-ray diffraction patterns from skinned rabbit psoas muscle indeed showed differences, mainly in the meridional reflections, but little difference in the off-meridional reflections. Molecular modeling is in progress to account for the observed changes. First indications are that the results are consistent with the recent EM result of Milligan et al. (Nature, 1995) which reported ADP induced movement, though only in smooth muscle myosin. Our findings of an additional structural state of strongly bound cross-bridges, particularly in the functioning skeletal muscle fibers, may provide the long sought evidence for structural change involved in the 'power stroke' generated by the actomyosin motor. X-ray diffraction is also applied to the problem of regulation of skeletal muscle contraction. Preliminary success has been achieved in exchanging endogenous troponin (Tn) with exogenous troponin following a protocol developed by Chalovich and Brenner (private communication). To improve the extent of exchange, the mechanism of exchange is being studied using fluorescence spectroscopy and other techniques in collaboration with Dr. Chalovich. Preliminary observations indicate that the binding of rigor cross-bridges to actin facilitates the exchange process. We have also obtained X-ray diffraction patterns from skinned fibers in which the exchanged TnC has bound biotin and avidin such that the reflections from Tn were intensified. Observations of changes in various regions of the troponin complex in response to activation should be feasible in the near future. The structure of cross-bridges in the presence of MgAMP-PNP has been further characterized in FY97 by two-dimensional X-ray diffraction. In saturating [MgAMP-PNP], the diffraction patterns are hardly distinguishable from those in the presence of [MgATP]. This finding clarifies considerable confusion remaining in the muscle field as to the state of cross-bridges in the presence of MgAMP-PNP. The present evidence is strong that MgAMP-PNP should be used as a stable, non- hydrolyzeable ATP analog. N-phenylmaleimide (NPM) labels one of the most reactive sulfhydryl groups of myosin. It has been thought that cross-bridges thus modified are locked in a state only capable of binding to actin weakly. Our recent data showed that in a Mg-free, ATP-free solution, the NPM modified cross-bridges are capable of binding to actin strongly. The NPM modification, in parallel with other chemical modifications, would be useful in identifying residues in the myosin molecule essential for structural transitions in the actomyosin ATP hydrolysis cycles.